嗜水气单胞菌疫苗免疫效果研究

选取对鲤鱼毒力强的嗜水气单胞菌菌株制成灭活疫苗,研究疫苗的免疫保护效应.采用甲醛灭活制备疫苗,分别对鲤鱼进行注射和浸泡试验,测定白细胞吞噬活性、淋巴细胞转化能力、血清、皮肤黏液的抗体效价及活菌攻毒后的免疫保护率.结果表明,受免鱼各项指标均明显高于对照组,注射组免疫保护率达90%,其中浸泡组各项略低于注射组.嗜水气单胞菌灭活疫苗对鲤鱼有显著免疫保护效应,可作为预防细菌性败血症感染的疫苗.     

嗜水气单胞菌灭活苗对林蛙免疫效果的初步研究

用分离于患红腿病的长白山中国林蛙的嗜水气单胞菌,采用热灭活的方法制成嗜水气单胞菌灭活苗,通过不同的途径免疫接种林蛙,通过测定受免林蛙血清中凝集抗体效价,探讨林蛙对热灭活的嗜水气单胞菌菌苗的免疫应答状况.结果表明,受免林蛙血清中凝集抗体效价可达到27,明显高于对照组.通过检测免疫林蛙的免疫保护率发现受免林蛙中口服免疫组、浸泡免疫组、腹腔注射组、肌肉注射组的免疫保护力可达71%、76%、59%、47%.     

鳗弧菌和溶藻弧菌二联疫苗对大菱鲆的免疫效果

将鳗弧菌（Vibrio anguillarum）和溶藻弧菌（V.alginolyticus）分别用0.3%福尔马林灭活,制备成鳗弧菌和溶藻弧菌2种单苗,并将2种菌等量配制成二联疫苗,单次腹腔注射免疫大菱鲆（Scophtalmus maximus）.通过检测免疫后替代途径补体活力、溶菌酶活力、抗体凝集效价的变化并分别进行攻毒实验比较疫苗的免疫效果.结果显示,3种疫苗对所测定的各免疫指标都有一定的促进作用,但二联疫苗后期效果低于2种单苗.二联疫苗对鳗弧菌和溶藻弧菌的免疫保护率分别为83.52%和83.24%,而鳗弧菌单苗对鳗弧菌攻毒、溶藻弧菌单苗对溶藻弧菌攻毒的免疫保护率分别为80.37%和74.89%.可以认为2株菌具有制备二联疫苗的可能性.[中国水产科学,2006,13（3）：397-402]     

A3α肽聚糖增强灭活嗜水气单胞菌疫苗免疫效果的研究

为了探讨 A_(3α)肽聚糖对灭活嗜水气单胞菌疫苗免疫效果的影响,用添加肽聚糖的饲料投喂注射接 种经甲醛灭活的嗜水气单胞菌菌苗的彭泽鲫,测定鲫鱼白细胞吞噬活性、血清和体表粘液溶菌酶活性、抗体 效价以及活菌攻毒后的免疫保护率。结果表明,肽聚糖与灭活菌苗联合使用,鱼体表粘液和血清溶菌酶活性、 白细胞吞噬活性、抗体效价以及活菌攻毒后的免疫保护率显著提高,表明 A_(3α)肽聚糖可增强灭活菌苗的免疫效 果。     

鳗弧菌灭活疫苗对牙鲆免疫效果的研究

制备鳗弧菌福尔马林灭活的全细胞疫苗，腹腔注射接种牙鲆，50d后进行第2次免疫。利用自制的兔抗鱼血清的抗血清通过ELISA实验检测了受免鱼特异性免疫应答水平，并通过攻毒实验，测定了疫苗对牙鲆的免疫保护率。结果表明，各免疫组血清抗体效价的最高值分别达1：512、1：2048、1：1024；各组受免鱼对人工攻毒均具有保护作用。用鳗弧菌攻毒后，免疫保护率分别达81．25％、87、50％、93．75％，而且发现受免鱼对副溶血弧菌(Vibrio parahaemolyticus)也表现了一定的交叉保护性，免疫保护率分别为46．15％、53．85％、53．85％。     

鳗弧菌三价灭活疫苗的长期免疫保护效果

鳗弧菌(Vibrio anguillarum)是一种能感染多种鱼类的条件致病菌,引起高致死率出血性败血病,流行于我国海水养殖环境,造成极大的经济损失。为此,本研究以鳗弧菌O1、O2和O3血清型菌株为抗原,制备了鳗弧菌三价灭活疫苗,以腹腔注射途径对健康大菱鲆(Scophthalmus maximus)(80.2±4.7)g进行一次免疫,在一次免疫后60 d以同等剂量和途径进行二次免疫。对一免组进行150 d的观测,结果显示,大菱鲆血清的3种抗原的特异抗体水平在免疫后14 d显著升高(P0.05),免疫后28 d达到最大值1∶320,免疫后28~150 d稳定在1∶106.7~1∶320,在免疫后14~150 d血清抗体效价均显著高于对照组(P0.05);相对免疫保护率(RPS)的检测结果显示,免疫后7 d大菱鲆抵抗鳗弧菌3种血清型病原感染的RPS分别为43.8%、38.9%和16.7%,免疫后28 d RPS均达最大值100%,免疫后28~120 d的RPS值保持在70%~100%,免疫后150 d的RPS值为35%~100%。对二免大菱鲆观测了90 d,二免后3~60 d的大菱鲆血清抗体水平显著高于同期一免的大菱鲆(P0.05),二免后60~90 d抗体水平下降,与同期一免鱼无显著差异(P0.05);二免大菱鲆的RPS值均高于70%,高于同期一免大菱鲆。上述结果显示,以鳗弧菌三价灭活疫苗一次免疫大菱鲆,抗体持续期不少于150 d,有效免疫保护期(RPS70%)不少于120 d;二次免疫大菱鲆,抗体持续期和有效免疫保护期(RPS70%)均不少于150 d。空白组最终体重略高于免疫组,但2组的特定生长率(SGR)无明显差异,说明三价疫苗对大菱鲆生长没有影响。     

鱼肠道弧菌外膜蛋白疫苗对牙鲆免疫效果的初步研究

为研究鱼肠道弧菌外膜蛋白疫苗对牙鲆的免疫效果,用该种疫苗对牙鲆进行了免疫试验.给牙鲆注射鱼肠道弧菌外膜蛋白疫苗,饲养25 d后分别用鱼肠道弧菌、鳗弧菌和溶藻弧菌进行攻毒,20 d后分别统计各组鱼的免疫保护率.结果表明,该疫苗对鱼肠道弧菌、鳗弧菌和溶藻弧菌的免疫保护率分别为84.6%、30.8%、33.3%.     

嗜水气单胞菌灭活疫苗免疫施氏鲟后的免疫应答反应与保护效果

为了研究嗜水气单胞菌灭活疫苗免疫施氏鲟(Acipenser schrenckii Brandt,1869)后的免疫应答反应与保护效果,本研究采用福尔马林灭活法制备嗜水气单胞菌灭活疫苗。通过腹腔注射途径免疫健康施氏鲟,对施氏鲟外周血的红细胞和白细胞数量、白细胞组成、吞噬活性、吞噬指数、血清酸性磷酸酶活性、血清溶菌酶活性、血清中和抗体效价等免疫指标及疫苗保护效果进行测定。结果显示,免疫组红、白细胞数量迅速上升,于免疫后第4天达峰值,分别为(8.50±0.17)×10~8个/mL和(8.96±0.44)×10~6个/mL;单核细胞和嗜中性粒细胞分类百分比于第7天达峰值,分别为10.50%和15.53%,极显著高于对照组(P0.01),淋巴细胞分类百分比在第21天才达峰值73.51%;吞噬指数和吞噬百分比均于第4天达到最高值,分别为4.53和30%;血清中溶菌酶活性和酸性磷酸酶活性分别在免疫后第7天和第14天达到峰值,极显著高于对照组(P0.01);血清抗体效价于免疫后第21天达到峰值1:203,极显著高于对照组(P0.01)。攻毒感染实验结果表明,免疫组的相对免疫保护率为76.84%。由此可见,嗜水气单胞菌灭活疫苗免疫施氏鲟后,能够显著诱导施氏鲟体内的免疫应答反应,增强鱼体的免疫保护能力。本研究为养殖鲟因嗜水气单胞菌感染引起疾病的免疫预防技术研究奠定了前期基础。     

单壁碳纳米管载嗜水气单胞菌外膜蛋白亚单位疫苗的研制及免疫效果评价

为了筛选细菌的疫苗候选蛋白,本实验根据课题组前期的研究,挑选了2个嗜水气单胞菌外膜蛋白(A0KLQ6和A0KH89),以A0KHR9(OmpAII)为对照,通过比较外膜蛋白与碳纳米管载外膜蛋白通过腹腔注射与浸泡免疫模式,比较和评价斑马鱼的免疫反应与免疫保护效果。结果显示,腹腔注射组和浸泡组与普通蛋白组对比,碳纳米管载蛋白能够显著提高斑马鱼免疫相关基因表达量。腹腔注射组在5μg剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得61.60%、 65.39%和84.96%相对免疫保护率(relative immune protection rate,RPS),在5μg剂量下免疫碳纳米管载蛋白SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0KH89分别获得68.10%、77.50%和90.43%的RPSs。浸泡组在40 mg/L剂量下免疫A0KHR9、A0KLQ6和A0KH89蛋白分别获得30.80%、25.85%和37.80%的RPSs,在40 mg/L最高剂量下免疫碳纳米管载蛋白疫苗SWCNTs-A0KHR9、SWCNTsA0KLQ6和SWCNTs-A0KH89分别获得63.60%、73.74%和68.49%的RPSs。研究表明,铁离子限制下嗜水气单胞菌外膜蛋白A0KLQ6和A0KH89等能够作为抗嗜水气单胞菌的亚单位疫苗候选蛋白,而单壁碳纳米管载蛋白通过注射免疫与浸泡免疫均能显著提升免疫疗效,是一种疫苗的适宜纳米载体。以上研究为寻找高效的嗜水气单胞菌亚单位疫苗候选蛋白及佐剂提供了理论依据。     

黄芪多糖佐剂对大菱鲆五联疫苗免疫增效作用

用福尔马林灭活哈维氏弧菌、鳗弧菌、迟钝爱德华氏菌、大菱鲆弧菌和溶藻胶弧菌制备成五联灭活疫苗,添加黄芪多糖作为佐剂。分别用添加黄芪多糖佐剂组和无佐剂组对大菱鲆进行腹腔注射,在第7、14、21、28、35、42、56天采取血样并检测抗体效价、溶菌酶活力和SOD活力,第56天进行人工攻毒实验,测定免疫保护力。结果表明,黄芪多糖佐剂组(免疫组2)和无佐剂组(免疫组1)两组的溶菌酶活力和SOD活力都在第28天达到最高值,且黄芪多糖佐剂组显著高于无佐剂组(P<0.05)。两个免疫组抗体效价也都在第28天达到最大值,但黄芪多糖佐剂组要显著高于无佐剂组(P<0.05),其中免疫组2的哈维氏弧菌、鳗弧菌、迟钝爱德华氏菌、大菱鲆弧菌和溶藻胶弧菌的抗体效价分别为27.25、26.75、28.5、28.75、26.25,免疫组1分别为26、25.75、26.75、27.5、25。用上述5种细菌分别进行人工攻毒实验,结果表明,免疫组2的免疫保护力依次为50.0%、50.0%、71.4%、62.5%、44.4%;免疫组1的免疫保护力依次为37.5%、30.0%、42.9%、50.0%、33.3%。实验结果证明,五联疫苗对大菱鲆具有免疫作用,而且添加黄芪多糖佐剂组的免疫效果显著好于无佐剂组。     

一株产絮凝剂芽孢杆菌的分离鉴定及絮凝剂特性分析

从山东及河北沿海分离到一株产絮凝剂细菌(编号200903091102,简称1102),分别用细菌全脂肪酸气相色谱法和16S rDNA序列分析比对法对该菌进行鉴定,两种方法的鉴定结果均显示细菌1102为一种芽孢杆菌(Bacillus sp.)。系统发育分析显示,细菌1102与枯草芽孢杆菌(B.subtilis)、特基拉芽孢杆菌(B.tequilensis)等亲缘关系最近。应用高氏1号培养基培养细菌1102,提取其絮凝剂进行絮凝力测定,结果显示,该菌所产絮凝剂的絮凝率达到80.19%。发酵72 h时,絮凝剂得率最高达19.5 g/L;分别采用凝胶渗透色谱法、苯酚-硫酸法及氨基酸自动分析仪对所得絮凝剂的相对分子质量、多糖含量及氨基酸含量进行分析,结果显示絮凝剂的重均相对分子质量为7 063 D,多糖质量分数占58.58%,氨基酸质量分数占2.49%。提示该菌在海水养殖中将具有较高的开发价值。本研究旨在为海水养殖废水的生物净化提供科学依据。     

Lactic acid bacteria in fish: a review

Fish are continuously exposed to a wide range of microorganisms present in the environment, and the microbiota of fish have been the subject of several reviews. This review evaluates lactic acid bacteria in fish, and focuses on the several investigations that have demonstrated that Streptococcus, Leuconostoc, Lactobacillus, and Carnobacterium belong to the normal microbiota of the gastrointestinal tract in healthy fish. However, it is well known that the population level of lactic acid bacteria associated with the digestive tract is affected by nutritional and environmental factors like dietary polyunsaturated fatty acids, chromic oxide, stress and salinity. Pathogenic lactic acid bacteria such as Streptococcus, Enterococcus, Lactobacillus, Carnobacterium and Lactococcus have been detected from ascites, kidney, liver, heart and spleen. Some antibiotic treatments and vaccinations have been proposed to cure or prevent these diseases that seem, however, to spread with the development of fish culture. It has also been reported that some lactic acid bacteria isolated from the gastrointestinal tract of fish can act as probiotics. These candidates are able to colonise the gut, and act antagonistic against Gram-negative fish pathogens. These harmless bacteriocin-producing strains may reduce the need to use antibiotics in future aquaculture.     

去除鱼类土腥味养殖系统的构建技术研究

以团头鲂、鲫鱼为试验对象,构建了一种基于人工生态系统的循环水养殖系统。通过底层陶粒吸附细菌、流水养鱼加快鱼类的代谢、湿地去除氨氮提高鱼的活性、生化箱培养硝化菌和反硝化菌来调控水质并起到控制水流速度的作用等技术手段来去除鱼类土腥味,同时研究不同盐度对其调控效果的影响。结果表明:本养殖系统对水体中的藻类和放线菌以及鱼体中的放线菌有明显的抑制作用。把(土池)池塘内养殖的鱼放入系统中暂养,一个月后土腥味显著降低,3个月后土腥味几乎消失;且随着水体盐度的升高,效果越加明显。     

Prebiotics in aquaculture: a review

A prebiotic is a non‐digestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or the activity of one or a limited number of bacteria in the colon. Despite the potential benefits to health and performance as noted in various terrestrial animals, the use of prebiotics in the farming of fish and shellfish has been less investigated. The studies of prebiotics in fish and shellfish have investigated the following parameters: effect on growth, feed conversion, gut microbiota, cell damage/morphology, resistance against pathogenic bacteria and innate immune parameters such as alternative complement activity (ACH50), lysozyme activity, natural haemagglutination activity, respiratory burst, superoxide dismutase activity and phagocytic activity. This review discusses the results from these studies and the methods used. If the use of prebiotics leads to health responses becoming more clearly manifested in fish and shellfish, then prebiotics might have the potential to increase the efficiency and sustainability of aquaculture production. However, large gaps of knowledge exist. To fully conclude on the effects of adding prebiotics in fish diets, more research efforts are needed to provide the aquaculture industry, the scientific community, the regulatory bodies and the general public with the necessary information and tools.     

Food safety hazards and the application of the principles of the hazard analysis and critical control point (HACCP) system for their control in aquaculture production

During the past 20 years there has been a dramatic global expansion in fish farming, with both positive and negative consequences. Although commercial aquaculture has contributed positively to the economies of many producing countries, there are considerable negative environmental and social impacts. In intensive and semi-intensive systems, artificial feeds supplemented with antibiotics are used to prevent the spread of disease and to improve feed conversion ratios. Current knowledge of the health and environmental impact of antibiotics used in aquaculture is poor, particularly in tropical regions. Residues may remain in fish used for human consumption and antibiotics released into the environment can lead to the development of antibiotic-resistant bacteria in the food chain. The accumulation of waste feeds in ponds stimulates the growth of bacteria, including human pathogens, which can contaminate products and lead to foodborne disease and the rejection of products in export markets. In extensive systems, where fish are produced mainly for the domestic market, different food safety concerns exist. The consumption of aquatic plants and raw or partially cooked freshwater fish has been associated with foodborne trematode infections. These are a major public health problem in East and South East Asia and occur when products that are contaminated by the infective stages of the parasites are consumed. This paper reviews food safety hazards associated with products from aquaculture and proposes the application of principles of the hazard analysis critical control point (HACCP) system as a general strategy to control the hazards identified.     

Simultaneous occurrence of Vibrio salmonicida and antibiotic-resistant bacteria in sediments at abandoned aquaculture sites

Abstract. The present authors have examined the survival of the fish pathogenic bacterium Vibrio salmonidda and the occurrence of antibiotic resistant bacteria in sediments from abandoned aquaculture sites. Survival of V. salmonidda was determined by fluorescent monoclonal antibody labelling combined with 4',6-diamidino-2-phenylindole (DAPI) staining. Using this method, the authors were able to detect V. salmonidda in sediment samples from six out of 12 abandoned fish farm locations. One out of the six positive stations was abandoned in 1982. Vibrio salmonidda was not detected at the three stations not affected by aquaculture (control locations) or in sediment samples taken 200–250m away from aqua-culture sites. When applying high-performance liquid chromatography, oxytctracyclinc or furazolidonc residues were not detected in sediment samples from control or abandoned aquaculture sites. Nevertheless, the percentage of bacteria resistant to oxytctracycline was higher in aquaculture sediments, as compared to control sediments. The simultaneous occurrence of fish pathogens and high levels of resistant bacteria in sediments increases the risk of transfer of resistance to the pathogens.     

Salmon farming and the environment

Abstract. The growth of the aquaculture industry and the general awareness of the environment have increased the concern of' the environmental impact from aquaculture.
As fish farms are open systems, all material that is not harvested as fish is released to the environment. The most important effluents are solid particles like feed and faeces, dissolved substances like nutrients and excretion products, and antibacterial agents.
The effect of these substances varies with their nature and quantity and with local conditions. Production and biomass of phytoplankton seem to be little influenced. The benthic community beneath and close to the cages is often altered. At poorly flushed sites the sea-bed may be anoxic and methane and hydrogensulphide are released from the sediments. Wild fauna, like fish, otters and birds are attracted to the farms.
The respiration and excretion of the fish directly influence water quality. Usually the oxygen tension is somewhat reduced in the net cages, while the concentration of ammonia is elevated in the net. Such conditions are assumed to influence fish growth and health.
Most of the antibiotic agents used in fish farming are persistent and may remain in high concentrations in the sediments up to one year after medication. The frequency of resistant bacteria in the sea-bed is normally increased, and there is a temporary drop in sediment metabolism after medication. Wild fish have been found to have high residues of antibiotic agents.
The organic loading of the sea-bed, the evolution of resistant bacteria and residues in the wild flsh are considered to be the most severe environmental impact of aquaculture. These are all associated with effluent of large particles, and thus mainly restricted to the site area.     

Microbial Ecology of the Gastrointestinal Tract of Fish and the Potential Application of Prebiotics and Probiotics in Finfish Aquaculture

Aquaculture is one of the fastest growing industries in the world. The need for enhanced disease resistance, feed efficiency, and growth performance of cultured organisms is substantial for various sectors of this industry. If growth performance and feed efficiency are increased in commercial aquaculture, then the costs of production are likely to be reduced. Also if more fish are able to resist disease and survive until they are of marketable size, the subsequent cost of medication and overall production costs would be reduced drastically. It has been documented in a number of food animals that gastrointestinal microbiota play important roles in affecting the nutrition and health of the host organism. Thus, various means of altering the intestinal microbiota to achieve favorable effects such as enhancing growth, digestion, immunity, and disease resistance of the host organism have been investigated in various terrestrial livestock as well as in humans. Dietary supplementation of prebiotcs, which are classified as non‐digestible food ingredients that beneficially affect the host by stimulating growth and/or activity of a limited number of health‐promoting bacteria such as Lactobacillus and Bifidobacter spp. in the intestine, while limiting potentially pathogenic bacteria such as Salmonella, Listeria and Escherichia coli, have been reported to favorably affect various terrestrial species; however, such information is extremely limited to date for aquatic organisms. Effects of probiotics, defined as live microbial feed supplements, on gastrointestinal microbiota have been studied in some fishes, but the primary application of microbial manipulations in aquaculture has been to alter the composition of the aquatic medium. In general, the gastrointestinal microbiota of fishes including those produced in aquaculture has been poorly characterized, especially the anaerobic microbiota. Therefore, more detailed studies of the microbial community of cultured fish are needed to potentially enhance the effectiveness of prebiotic and probiotic supplementation. This review summarizes and evaluates current knowledge of intestinal microbial ecology of fishes, the various functions of this intestinal microbial community, and the potential for further application of prebiotics and probiotics in aquaculture.     

益生菌在盐碱养殖水体中调控作用的研究进展

我国内陆、滨海地区有丰富且尚未有效开发利用的盐碱水资源,在盐碱水域进行水产养殖已成为其重要的开发利用途径之一,而盐碱水体的高pH、高盐、高碱特点严重限制了水产养殖对象的生长发育。益生菌具有净化水质、调节pH、降低氨氮和提高鱼类盐碱耐受能力等综合作用,是一种高效无污染的水质改良剂。合理使用益生菌可有效改良盐碱水体,提高水产养殖效益。文章综述了光合细菌、乳酸菌、芽孢杆菌、酵母菌等不同菌种制剂及其在适应、改良盐碱水体方面的研究进展。     

3种滤料生物滤器的挂膜与黑鲷幼鱼循环水养殖效果

采用微生态净水剂作为菌种,对3种生物滤料(陶环、弹性毛刷和爆炸棉)构建的生物滤器进行生物膜培养,并以3种生物滤器为基础构建简易循环水养殖系统,进行黑鲷(Sparus macrocephalus)幼鱼养殖实验。结果表明,陶环生物滤器、弹性毛刷生物滤器和爆炸棉生物滤器的生物膜成熟时间分别为25 d、32 d和28 d。黑鲷幼鱼经过40d的饲喂,3个实验组鱼的体质量与对照组相比,均差异显著(P<0.05),成活率均达到95%以上;实验组鱼血清溶菌酶(LSZ)活性和肝组织总超氧化物歧化酶(T-SOD)活性均显著高于对照组(P<0.05);实验组鱼体消化道内菌群数量及其多样性要明显高于对照组。结果表明,不同滤料在相同工况条件下挂膜成熟时间不同;微生态净水剂作为挂膜菌种效果良好;循环水系统养殖模式不仅可以提高鱼体的生长速度,还能增强鱼体免疫性能。